CN101935692A - Microsatellite marking method applicable to parentage determination of apostichopus japonicus - Google Patents
Microsatellite marking method applicable to parentage determination of apostichopus japonicus Download PDFInfo
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- CN101935692A CN101935692A CN2009102315753A CN200910231575A CN101935692A CN 101935692 A CN101935692 A CN 101935692A CN 2009102315753 A CN2009102315753 A CN 2009102315753A CN 200910231575 A CN200910231575 A CN 200910231575A CN 101935692 A CN101935692 A CN 101935692A
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Abstract
The invention belongs to the technical field of DNA marking in molecular biology and relates to a molecular assisted technology for genetic breeding, in particular to a microsatellite marking method applicable to parentage determination of apostichopus japonicus, which comprises four steps of sourcing microsatellite loci, designing a primer, optimizing the primer and establishing a microsatellite marker parentage determination system, and the specific process is as follows: firstly using an enhanced library-colony in-situ hybridization method to screen microsatellite sequences, and using microsatellite retrieval software to carry out rapid separation of microsatellite fragments; designing the primer on the microsatellite repeated flanking sequence, optimizing the primer and leading the primer to become a microsatellite marker; then carrying out comprehensive evaluation on repeatability, stability and polymorphism information content value during the amplification process of the microsatellite marker, determining the microsatellite markers therein as a microsatellite parentage determination system of the apostichopus japonicus, and being used for differentiation among different parentages of the apostichopus japonicus or identification and determination among individuals. The method can realize the effects of the markers and has the advantages of feasible design principle, simple marking way and good applicability.
Description
Technical field:
The invention belongs to molecular biology dna marker technical field, relate to a kind of molecule ancillary technique of genetic breeding, particularly a kind of microsatellite marker method that is applicable to that imitative stichopus japonicus family is identified.
Background technology:
It is the effective ways of rearing new variety that family is selected breeding, and to be undertaken that system selects be the important means of genetic breeding by setting up family, obtained extensive success in animals and plants.Complete, pedigree information can produce very big influence to breeding process accurately, can not accurately confirm the sibship between individuality, can cause the reduction of genetic progress in the breeding process.By paternity test correct decision parent, the pedigree information that corrects mistakes is to promoting breed improvement significant.Simultaneously, in manually putting in a suitable place to breed, releasing process, cause individual vitality to reduce, keep the genetic diversity of species, also need to understand fully the sibship of breeding parent for reducing inbreeding.Microsatellite DNA (microsatellite) claim simple repeated sequence again, since its have distribute wide, polymorphism is high, single locus gene be codominant inheritance, detection method fast and stable, required DNA amount less, be easy to advantage such as analysis, remarkable advantages is arranged in paternity test.Imitative stichopus japonicus (Apostichopus japonicus) is the famousst and precious a kind of in the beche-de-mer, and NATURAL DISTRIBUTION is in northern China, Japan, the Korea peninsula and Muscovite the Far East Area.Because the economic worth height, in recent years imitative stichopus japonicus propagate artificially and the propagation development of releasing swift and violent, become one of important sea farming kind of northern China.Utilize molecular marking technique, set up the microsatellite marker family identification system that a cover is applicable to imitative stichopus japonicus, to keeping family information in the seed selection, determine sibship, following the trail of family strong instrument is provided, for good basis is established in the breeding planning of imitative stichopus japonicus, it is the method that the scientific research personnel in present technique field makes every effort to probe into, but at present, the success of this method and application thereof do not see that as yet report is arranged.
Summary of the invention:
The objective of the invention is to overcome the shortcoming of prior art existence, seek to design the microsatellite marker family authentication method that a cover is applicable to imitative stichopus japonicus, be implemented in the mark basis of imitating maintenance family information in the stichopus japonicus seed selection process, determining sibship, tracking family.
To achieve these goals, the present invention includes four steps of establishment of microsatellite locus source, design of primers, primer optimization and microsatellite marker family identification system, utilize enriched library-bacterium colony in situ hybridization method screening microsatellite sequence earlier, use little satellite retrieval software RepeatReporter 1.5 and carry out the segmental sharp separation of little satellite; At little satellite repeated flanking sequences design primer, the one-step optimization primer of going forward side by side becomes microsatellite marker; Then repeatability, stability and the polymorphism information content values of microsatellite marker in amplification procedure carried out comprehensive evaluation, confirm 9 imitative little satellite identification systems of stichopus japonicus family (table 1) of microsatellite markers conduct wherein, be used for the differentiation between the different familys of imitative stichopus japonicus or carry out identification and evaluation between individuality, thereby realize the mark effect.Concrete steps are:
(1), microsatellite locus source: the genomic dna that extracts imitative stichopus japonicus, 4 ℃ of refrigerators are preserved standby, utilize the DNA that extracts then, obtain imitative stichopus japonicus genomic DNA fragment with existing restriction enzyme enzyme process, and the structure enriched library, then containing in the enriched library inserting the in situ hybridization of segmental intestinal bacteria bacterium colony, positive colony is determined in radioautograph then, the positive colony that checks order at last obtains containing little satellite multiple dna sequence dna;
(2), design of primers: utilize software PrimerPremier 5.0 and Oligo 6.44 design primers in little satellite repeated flanking sequences, the design of primers condition is: primer length is 19-25mer; GC content is 40%-60%; Annealing temperature is the 45-65 degree; Expection PCR product length is 100-400bp;
(3), primer optimization: different primers are according to different Tm values, at the enterprising trip temperature gradient optimizing of thermograde PCR instrument, respectively do 10 degree up and down in the Tm value, amplified reaction adopts BiometraT-Gradient PCR system, the PCR program is: 95 ℃ of sex change 45s, annealing 45s, 72 ℃ are extended 45s, 30 circulations are carried out in reaction, the preceding 95 ℃ of pre-sex change 5min that circulate first, and 72 ℃ are extended 5min again after the last loop ends, reaction system is 20 μ l, contain 1 * PCR reaction buffer, the dNTPs of 0.2mM, the Mg of 1.2mM
2+The positive and negative primer of each 0.2 μ M, the Taq archaeal dna polymerase of 1U, the template DNA of 80ng, this template is to appoint to get 5 DNA of individual balanced mix and obtain from 48 individualities, the PCR product that amplification obtains detects with polyacrylamide gel electrophoresis-EB coloring system of 10%, and choosing assorted temperature of answering with the PCR reaction pair less, that the brightness of specificity product is higher is the optimum annealing temperature Ta of this primer;
(4), the establishment of microsatellite marker family identification system: the available polymorphism information content value of the polymorphism level of microsatellite locus (Polymorphism Information Content, PIC) weigh, generally speaking, the polymorphism information content value can reflect that some genetic markers comprise or the capacity of the genetic information that can provide, when PIC＞0.5, show that this genetic marker can provide prolific hereditary information; When 0.25＜PIC＜0.5, show that this genetic marker can comparatively reasonably provide genetic information; And when PIC＜0.25, show that the available genetic information of this genetic marker is relatively poor, Ta value according to above-mentioned optimization acquisition, choose 48 individualities carry out polymorphism information content values as colony calculating, the PCR program is 95 ℃ of sex change 45s, the optimum annealing temperature Ta annealing 45s that each primer is optimized, 72 ℃ are extended 45s, and 30 circulations are carried out in reaction; Preceding 95 ℃ of pre-sex change 5min first circulate; 72 ℃ are extended 5min again after the last loop ends, and reaction system is 20 μ l, contains 1 * PCR reaction buffer, the dNTPs of 0.2mM, the Mg of 1.2mM
2+The positive and negative primer of each 0.2 μ M, the Taq archaeal dna polymerase of 1U, the template DNA of 20ng, pcr amplification product is through 10% native polyacrylamide gel electrophoresis, voltage is 5V/cm, and electrophoresis finishes after EB (concentration is 0.15 μ g/ml) dyeing, and the ultraviolet visualization imaging also utilizes formula PIC=1-∑ Pi to the electrophoresis band spectrum
2Calculate, wherein Pi is an i allelic frequency, the gene frequency of all sites is calculated by software POPGENE32, according to the calculation result of PIC value, remove repeatability, poor stability, PIC is less than 0.25 mark, final screening obtains repeatability, good stability, the PIC value is greater than 9 in 0.25 site, and as apostichopus japonicus microsatellite mark family identification system, the family that is used for imitative stichopus japonicus is distinguished and individual recognition.
The present invention compared with prior art, its principle of design is feasible, mark mode is simple, applicability is good, is adapted to the microsatellite marker that imitative stichopus japonicus family is identified especially.
Description of drawings:
Fig. 1 is that the site PSC2 of the inventive method is to female (♀) and male (♂) parent and 5 " filial generation " amplification synoptic diagram (1-5) thereof in the imitative stichopus japonicus 1# family, pcr amplification by this microsatellite locus, No. 5 individuality is identified the offspring who is not these two parents, because it does not meet the Mendelian inheritance law of segregation of parent in this site, by the analysis-by-synthesis of a plurality of microsatellite locus, can effectively discern the sibship of identifying between individuality.
Embodiment:
Below by embodiment the present invention is described in detail.
Present embodiment comprises following four steps:
(1), microsatellite locus source: the genomic dna that extracts imitative stichopus japonicus, 4 ℃ of refrigerators are preserved standby, utilize the DNA that extracts then, obtain imitative stichopus japonicus genomic DNA fragment with existing restriction enzyme enzyme process, and the structure enriched library, then containing in the enriched library inserting the in situ hybridization of segmental intestinal bacteria bacterium colony, positive colony is determined in radioautograph then, the positive colony that checks order at last obtains containing little satellite multiple dna sequence dna;
(2), design of primers: utilize software PrimerPremier 5.0 and Oligo 6.44 design primers in little satellite repeated flanking sequences, the design of primers condition is: primer length is 19-25mer; GC content 40%-60%; Annealing temperature 45-65 degree; Expection PCR product length is 100-400bp;
(3), the optimization of primer: different primers are according to different Tm values, the enterprising trip temperature gradient optimizing of thermograde PCR instrument (respectively doing 10 degree up and down) in the Tm value, amplified reaction adopts Biometra T-Gradient PCR system, the PCR program is: 95 ℃ of sex change 45s, annealing 45s, 72 ℃ are extended 45s, 30 circulations are carried out in reaction, preceding 95 ℃ of pre-sex change 5min first circulate, 72 ℃ are extended 5min again after the last loop ends, and reaction system is 20 μ l, contains 1 * PCR reaction buffer, 0.2mM dNTPs, the Mg of 1.2mM
2+The positive and negative primer of each 0.2 μ M, the Taq archaeal dna polymerase of 1U, the template DNA of 80ng, this template is to appoint to get 5 DNA of individual balanced mix and obtain from 48 individualities, the PCR product that amplification obtains detects with polyacrylamide gel electrophoresis-EB coloring system of 10%, and choosing assorted temperature of answering with the PCR reaction pair less, that the brightness of specificity product is higher is the optimum annealing temperature Ta of this primer;
(4), the establishment of the microsatellite marker family identification system of imitative stichopus japonicus: available polymorphism information content value (the Polymorphism InformationContent of the polymorphism level of microsatellite locus, PIC) weigh, generally speaking, the polymorphism information content value can reflect that some genetic markers comprise or the capacity of the genetic information that can provide, when PIC＞0.5, show that this genetic marker can provide prolific hereditary information; When 0.25＜PIC＜0.5, show that this genetic marker can comparatively reasonably provide genetic information; And when PIC＜0.25, show that the available genetic information of this genetic marker is relatively poor, Ta value according to above-mentioned optimization acquisition, choose 48 individualities carry out polymorphism information content values as colony calculating, the PCR program is 95 ℃ of sex change 45s, Ta (optimum annealing temperature that each primer is optimized) annealing 45s, 72 ℃ are extended 45s, and 30 circulations are carried out in reaction; Preceding 95 ℃ of pre-sex change 5min first circulate; 72 ℃ are extended 5min again after the last loop ends, and reaction system is 20 μ l, contains 1 * PCR reaction buffer, the dNTPs of 0.2mM, the Mg of 1.2mM
2+The positive and negative primer of each 0.2 μ M, the Taq archaeal dna polymerase of 1U, the template DNA of 20ng, pcr amplification product is through 10% native polyacrylamide gel electrophoresis, voltage is 5V/cm, and electrophoresis finishes after EB (concentration is 0.15 μ g/ml) dyeing, and the ultraviolet visualization imaging also utilizes formula PIC=1-∑ Pi to the electrophoresis band spectrum
2Calculate, wherein Pi is an i allelic frequency, the gene frequency of all sites is calculated by software POPGENE32, according to the calculation result of PIC value, remove repeatability, poor stability, PIC is less than 0.25 mark, final screening obtains repeatability, good stability, the PIC value is greater than 9 in 0.25 site, and as apostichopus japonicus microsatellite mark family identification system, the family that is used for imitative stichopus japonicus is distinguished and individual recognition.
Embodiment:
Present embodiment is divided into extracting imitates stichopus japonicus DNA, pcr amplification and three step contents of electrophoresis detection PCR reaction conditions:
Extract imitative stichopus japonicus DNA: the imitative stichopus japonicus family of selecting for use is picked up from certain plant in marine site, Rongcheng, Shandong, in the 1# family, get female and the male parent, " filial generation " of 48 health of picked at random is individual as experiment material again, get the about 200mg of muscle tissue after the imitative stichopus japonicus vivisection, add 500 μ l CTAB lysate [EDTA:200mM; Tris-Cl:100mM, PH=8.0; NaCl:1.4M; CTAB:2% (W/V); Add 1.5% beta-mercaptoethanol before using], shred, 60 ℃ of processing up to the lysate clarification, add the saturated phenol of equal-volume (250 μ l), chloroform/primary isoamyl alcohol (24: 1) (250 μ l), extracting 3 times; Get supernatant liquor, add equal-volume chloroform/primary isoamyl alcohol (500 μ l) extracting 1 time, get supernatant liquor, add 50 μ l NaAc (3M), slowly shake up, fill it up with the ice dehydrated alcohol, 12,000 left the heart 10 minutes, nucleic acid is deposited in the pipe end, 70% ethanol (1000 μ l) washing precipitation and drying are all volatilized up to ethanol, add sterilized water and a small amount of RNase A of 100 μ l, and 4 ℃ all dissolve up to DNA;
Pcr amplification: the PCR reaction system is 20 μ l, contains the imitative stichopus japonicus genomic dna of 40ng, the primer of 0.2mmol/l, the dNTPs of 200mmol/l, the Mg of 200mmol/l
2+, 1 * PCR reaction buffer, the Taq archaeal dna polymerase of 1U, the PCR program parameter is: 95 ℃ of sex change 45s, Ta 45s that anneals, 72 ℃ are extended 45s, and 30 circulations are carried out in reaction, and preceding 95 ℃ of pre-sex change 5min first circulate; 72 ℃ are extended 5min, 4 ℃ of preservations again after the last loop ends;
Electrophoresis detection PCR reaction conditions: pcr amplification product is through 10% native polyacrylamide gel electrophoresis, voltage is 5V/cm, comprise two molecular weight standards---pUC19/HaeIII on every glue, dye with ethidium bromide (concentration 0.15mg/mL) after electrophoresis 2-3 hour, ultraviolet imagery and electrophoretic band analyzed on the gel imaging system, it is big or small accurately definite to utilize software Quantity One that each individual amplified production is carried out, thereby carrying out fast and accurately genotype determines, according to mendel's law the genotype that obtains is analyzed, carrying out family identifies and the sibship analysis, with of the foundation of 9 microsatellite markers, for the family breeding of imitative stichopus japonicus is had laid a good foundation as imitative stichopus japonicus family identification system.
Claims (1)
1. one kind is applicable to the microsatellite marker method of imitating the evaluation of stichopus japonicus family, comprises four steps of establishment of microsatellite locus source, design of primers, primer optimization and microsatellite marker family identification system, it is characterized in that:
(1), microsatellite locus source: the genomic dna that extracts imitative stichopus japonicus, 4 ℃ of refrigerators are preserved standby, utilize the DNA that extracts then, obtain imitative stichopus japonicus genomic DNA fragment with existing restriction enzyme enzyme process, and the structure enriched library, containing in the enriched library inserted the in situ hybridization of segmental intestinal bacteria bacterium colony, positive colony is determined in radioautograph then, the positive colony that checks order at last obtains containing little satellite multiple dna sequence dna;
(2), design of primers: utilize software PrimerPremier 5.0 and Oligo 6.44 design primers in little satellite repeated flanking sequences, the design of primers condition is: primer length is 19-25mer; GC content is 40%-60%; Annealing temperature is the 45-65 degree; Expection PCR product length is 100-400bp;
(3), primer optimization: different primers are according to different Tm values, at the enterprising trip temperature gradient optimizing of thermograde PCR instrument, respectively do 10 degree up and down in the Tm value, amplified reaction adopts BiometraT-Gradient PCR system, the PCR program is: 95 ℃ of sex change 45s, annealing 45s, 72 ℃ are extended 45s, 30 circulations are carried out in reaction, the preceding 95 ℃ of pre-sex change 5min that circulate first, and 72 ℃ are extended 5min again after the last loop ends, reaction system is 20 μ l, contain 1 * PCR reaction buffer, the dNTPs of 0.2mM, the Mg of 1.2mM
2+The positive and negative primer of each 0.2 μ M, the Taq archaeal dna polymerase of 1U, the template DNA of 80ng, this template is to appoint to get 5 DNA of individual balanced mix and obtain from 48 individualities, the PCR product that amplification obtains detects with polyacrylamide gel electrophoresis-EB coloring system of 10%, and the temperature that choosing mixes is with less, the high PCR reaction pair of specificity product brightness is answered is the annealing temperature Ta of this primer;
(4), the establishment of microsatellite marker family identification system: the polymorphism level of microsatellite locus is weighed with the polymorphism information content value, the polymorphism information content value reflect that genetic marker comprises or the capacity of the genetic information that can provide, when PIC＞0.5, show that this genetic marker provides genetic information; When 0.25＜PIC＜0.5, show that this genetic marker can rationally provide genetic information; And when PIC＜0.25, show that the genetic information that this genetic marker provides is poor, Ta value according to above-mentioned optimization acquisition, choose 48 individualities carry out polymorphism information content values as colony calculating, the PCR program is 95 ℃ of sex change 45s, the annealing temperature Ta annealing 45s that each primer is optimized, 72 ℃ are extended 45s, and 30 circulations are carried out in reaction; Preceding 95 ℃ of pre-sex change 5min first circulate; 72 ℃ are extended 5min again after the last loop ends, and reaction system is 20 μ l, contains 1 * PCR reaction buffer, the dNTPs of 0.2mM, the Mg of 1.2mM
2+The positive and negative primer of each 0.2 μ M, the Taq archaeal dna polymerase of 1U, the template DNA of 20ng, pcr amplification product is through 10% native polyacrylamide gel electrophoresis, voltage is 5V/cm, and electrophoresis finishes after concentration is the EB dyeing of 0.15 μ g/ml, and the ultraviolet visualization imaging also utilizes formula PIC=1-∑ Pi to the electrophoresis band spectrum
2Calculate, wherein Pi is an i allelic frequency, the gene frequency of all sites is calculated by software POPGENE32, according to the calculation result of PIC value, remove repeatability, poor stability, PIC is less than 0.25 mark, screening obtains repeatability, good stability, the PIC value as apostichopus japonicus microsatellite mark family identification system, realizes that the family of imitative stichopus japonicus is distinguished and individual recognition greater than 0.25 site.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653785A (en) * | 2011-03-03 | 2012-09-05 | 华中农业大学 | Identifying method of megalobrama amblycephala family by microsatellite |
| CN113789391A (en) * | 2021-07-07 | 2021-12-14 | 中国海洋大学 | Apostichopus japonicus breeding whole genome 50K SNP chip and application |
| CN117004701A (en) * | 2023-09-27 | 2023-11-07 | 中国科学院海洋研究所 | Molecular marker for sex identification of apostichopus japonicus and application |
-
2009
- 2009-12-03 CN CN2009102315753A patent/CN101935692A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653785A (en) * | 2011-03-03 | 2012-09-05 | 华中农业大学 | Identifying method of megalobrama amblycephala family by microsatellite |
| CN102653785B (en) * | 2011-03-03 | 2013-10-23 | 华中农业大学 | Identifying method of megalobrama amblycephala family by microsatellite |
| CN113789391A (en) * | 2021-07-07 | 2021-12-14 | 中国海洋大学 | Apostichopus japonicus breeding whole genome 50K SNP chip and application |
| CN117004701A (en) * | 2023-09-27 | 2023-11-07 | 中国科学院海洋研究所 | Molecular marker for sex identification of apostichopus japonicus and application |
| CN117004701B (en) * | 2023-09-27 | 2024-01-02 | 中国科学院海洋研究所 | Molecular marker for sex identification of apostichopus japonicus and application |
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Application publication date: 20110105 |